EP1851309A2 - Mutants de acyltransferase de isopenicillin n - Google Patents

Mutants de acyltransferase de isopenicillin n

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Publication number
EP1851309A2
EP1851309A2 EP06708501A EP06708501A EP1851309A2 EP 1851309 A2 EP1851309 A2 EP 1851309A2 EP 06708501 A EP06708501 A EP 06708501A EP 06708501 A EP06708501 A EP 06708501A EP 1851309 A2 EP1851309 A2 EP 1851309A2
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EP
European Patent Office
Prior art keywords
seq
polypeptide
acid sequence
amino acid
acyltransferase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP06708501A
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German (de)
English (en)
Inventor
Anthony Cox
Anke Krebber
Roelof Ary Lans Bovenberg
Yong Hong Chen
Stephane J. Jenne
Matthew Tobin
Charlene To La Ching
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DSM IP Assets BV
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DSM IP Assets BV
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Priority to EP06708501A priority Critical patent/EP1851309A2/fr
Publication of EP1851309A2 publication Critical patent/EP1851309A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01164—Isopenicillin-N N-acyltransferase (2.3.1.164)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin

Definitions

  • the present invention relates generally to novel acyltransferase polypeptides that are useful in the production of beta-lactam intermediates and antibiotic compounds.
  • beta-lactam antibiotics The most important classes of the beta-lactam antibiotics are the penicillins (penams) and the cephalosporins (ceph-3-ems). Penicillins are produced by filamentous fungi only (Penicillium chrysogenum or Aspergillus nidulans), whereas cephalosporins are produced by filamentous fungi (Acremonium chrysogenum) as well as bacteria (e.g. Streptomyces clavuligerus). All beta-lactam antibiotics share the common structural feature of a four-member beta-lactam ring.
  • the naturally occuring penams and ceph-3-ems are synthesized from the same three amino acid precursors: L- alpha-aminoadipic acid (L-alpha-AAA), L-cysteine, and L-valine.
  • L-alpha-AAA L- alpha-aminoadipic acid
  • L-cysteine L-cysteine
  • L-valine L-valine
  • the first two steps in the biosynthesis pathways of penicillins and cepalosporins are the same.
  • the amino acid precursors are condensed to the tripeptide delta-L-alpha- aminoadipyl-L-cysteinyl-D-valine (ACV).
  • ACCV tripeptide delta-L-alpha- aminoadipyl-L-cysteinyl-D-valine
  • the requisite reaction cycle e.g., recognition, activation and formation of peptide bonds
  • ACVS multifunctional enzyme ACV synthetase
  • the resulting linear tripeptide is cyclized by oxidative ring closure leading to the formation of a bicyclic ring structure, i. e., the four- member beta-lactam ring fused to the five-membered thiazolidine ring which is characteristic of all penams.
  • Cyclization is catalyzed by the enzyme isopenicillin N synthase (IPNS).
  • IPNS isopenicillin N synthase
  • the resulting compound represents the first bioactive intermediate and is referred to as isopenicillin N (IPN). See generally, e.g., EP 0 422 790 (Miller et al.). After IPN synthesis, the biosynthetic pathways leading to the production of the penicillins and cephalosporins diverge.
  • the hydrophilic L-alpha-AAA side chain of IPN can be replaced by a hydrophobic acyl group in a third and final exchange step.
  • the side chain may be of intracellular origin (e.g., hexonic acid or octenoic acid) or supplied exogenously.
  • penicillin V and penicillin G produced by adding the exogenous side chain precursors phenoxyacetic or phenylacetic acid, respectively.
  • the exchange is catalyzed by acyl coenzyme A (CoA):isopenicillin N acyltransf erase (AT) which is encoded by the penDE gene.
  • CoA acyl coenzyme A
  • AT isopenicillin N acyltransf erase
  • ceph-3-ems are produced by the isomerization of the L-alpha-
  • the alpha-aminoadipic acid side chain of IPN is isomerized to produce penicillin N, after which the five-membered thiazolidine ring of the penicillin is "expanded" by deacetoxycephalosporin synthetase (DAOCS) expandase activity to produce deacetoxycephalosporin C (DAOC) which comprises the six-membered dihydrothiazine ring that is characteristic of the cephalosporins.
  • DOCS deacetoxycephalosporin synthetase
  • beta-lactams e.g., penicillin F, isopenicillin N, cephalosporin C, etc.
  • many of the so-called natural beta-lactams are of limited utility as antibiotics because they are unstable, difficult to purify from fermentation broth, have only limited antibiotic effect, and/or are produced in low yield.
  • Replacing the side chains of these beta-lactams with other side chains leads to the formation of semisynthetic penicillins and cephalosporins, such as amoxycillin, ampicillin and cephalexin, which are more stable, easier to isolate, and have a higher antibiotic activity.
  • the large variety of side chains found in commercially significant beta-lactam compounds has placed increased importance on achieving more economic and efficient methods of preparing key intermediates for synthesis of various beta-lactam compounds.
  • cephalosporin intermediate 7-ADCA is an important intermediate for the synthesis of many semisynthetic cephalosporins. It is currently produced by either chemical derivatization of penicillin G or by a bioprocess as described in EP 0532341.
  • adipyl-7-ADCA is formed by a Penicillium chrysogenum strain modified to express expandase and fed with the side chain precursor adipic acid. Subsequent removal of the adipyl side chain with a suitable enzyme leads to formation of 7-ADCA.
  • the AT enzyme thus is capable of accepting other side chains than phenyl- or phenoxyacetic acid, it is unpredictable whether or not the capacity of the AT enzyme to accept other side chains than phenyl- or phenoxyacetic acid can be improved or its substrate specificity modified.
  • Figure 1 is a schematic of a 6343 bp vector (pET-penDE Pc) containing a T7 promoter, a T7 terminator, and a kanamycin resistance gene.
  • Figure 2 is an alignment display which provides the amino acid sequences of the acyltransferase enzymes disclosed herein.
  • the present invention provides polypeptides having acyltransferase activity with altered substrate specificity as compared to the native Penicillium chrysogenum acyltransferase.
  • the present invention provides acyltransferase polypeptides with an improved capacity as compared to the native P. chrysogenum acyltransferase to form adipoyl-6-APA from alpha-aminoadipoyl-6-APA. More particularly, said improved capacity is at least 1.5 times the activity of the native P. chrysogenum acyltransferase, preferably at least 2 times, more preferably at least 5 times, more preferably at least 10 times, most preferably at least 30 times.
  • the present invention is directed to an isolated polypeptide having acyltransferase activity, wherein the isolated polypeptide is selected from the group consisting of:
  • polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 52 (d) a polypeptide having an amino acid sequence that is at least 96% identical to SEQ ID NO: 54 and having at least one residue selected from the group consisting of Arg at position 97, VaI at position 221 , Pro at position 251 , GIu at position 254, Ne at position 259, GIy at position 277, GIu at position 291, and GIy at position 299; and (e) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 42 and SEQ ID NO: 52.
  • the present invention is directed to polynucleotides that encode acyltransferase polypeptides.
  • the present invention is directed to expression vectors, host cells, and methods that are useful for producing beta-lactam compounds using the acyltransferase polypeptides of the present invention.
  • the present invention provides novel polypeptides having acyltransferase ("AT”) activity selected from the group consisting of:
  • the present invention is directed to an isolated polypeptide having acyltransferase activity, wherein the isolated polypeptide is selected from the group consisting of:
  • a polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 52 (d) a polypeptide having an amino acid sequence that is at least 96% identical to SEQ ID NO: 54 and having at least one residue selected from the group consisting of Arg at position 97, VaI at position 221 , Pro at position 251 , GIu at position 254, Ne at position 259, GIy at position 277, GIu at position 291, and GIy at position 299; and (e) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 42 and SEQ ID NO: 52.
  • Acyltransferase polypeptides of the present invention are useful in the production of beta-lactam compounds, such as, for example, penicillin antibiotic compounds having adipoyl side chains.
  • acyltransferase polypeptide As used herein, the terms “acyltransferase polypeptide,” “AT polypeptide,” “acyltransferase enzyme,” and “AT enzyme” are used interchangeably herein to refer to a polypeptide that exhibits acyltransferase activity.
  • acyltransferase activity is used herein to refer to the ability of an enzyme to catalyze at least the conversion of 6-aminopenicillanic acid (“6-APA”) and adipoyl-coenzyme A ("Ad- CoA) to a detectable amount of adipoyl-6-aminopenicillanic acid (“Ad-6-APA”) using the assay described in Example 3.
  • Ad-6-APA is a useful intermediate that can be converted to a penicillin antibiotic having an adipoyl side chain or to the 6-APA nucleus.
  • ad-6-APA is a useful intermediate that can be expanded to adipoyl-7- ADCA as described in e.g. EP 0 532 341.
  • the AT enzyme is also able to catalyze the hydrolysis of IPN, whereby the alpha-AAA side chain is cleaved off, resulting in the formation of 6-APA.
  • acyltransferase polynucleotides and "AT polynucleotides” are used interchangeably herein to refer to polynucleotides that encode acyltransferase polypeptides. It is to be understood that the reference to adipoyl should not be considered as a limitation, but only as a means to characterize at least one of the catalytic capacities of the enzyme.
  • isolated refers to a polynucleotide or polypeptide that is substantially free of other material with which it is normally found in nature, and thus is substantially free of other naturally occurring cellular material, as well as culture medium.
  • isolated polynucleotide is largely free of sequences that flank the polynucleotide in its native genomic location.
  • polynucleotide refers toe DNA, RNA, or synthetic analogues thereof.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acids.
  • the present invention also provides acyltransferase polypeptides that have an amino acid sequence at least 97% identical to SEQ ID NO: 38, typically at least 98% identical to SEQ ID NO: 38, and in some embodiments, at least 99% identical to SEQ ID NO: 38.
  • the present invention provides acyltransferase polypeptides that have an amino acid sequence that is at least 96% identical to SEQ ID NO: 52.
  • the present invention includes AT polypeptides having an amino acid sequence that is at least 97% identical to SEQ ID NO: 52, as well as those that are at least 98% and at least 99% identical to SEQ ID NO: 52.
  • the present invention provides acyltransferase polypeptides that have an amino acid sequence that is at least 96% identical to wild type acyltransferase from P. chrysogenum (SEQ ID NO: 54), and which also has in its sequence, at least one residue selected from the group consisting of Arg at position 97, VaI at position 221, Pro at position 251 , GIu at position 254, Ne at position 259, GIy at position 277, GIu at position 291, and GIy at position 299.
  • the amino acid sequence of the AT polypeptide has at least two residues selected from this group, and sometimes at least four residues, often up to about six residues (e.g., Pro at position 251, GIu at position 254, Ne at position 259, GIy at position 277, GIu at position 291, and GIy at position 299).
  • the amino acid positions recited herein are the positions that correspond to SEQ ID NO: 54 upon optimal alignment of the AT polypeptide sequence with SEQ ID NO: 54.
  • optically aligned refers to the alignment created using the Clustal W algorithm (Nucleic Acid Research. 22(22): 4673-4680 (1994). With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds to" the position in the reference sequence with which the residue is paired in the alignment.
  • acyltransferase polypeptides of the present invention are provided that have higher acyltransferase activity than that of the wild type P. chrysogenum acyltransferase (SEQ ID NO: 54) as measured in the assay described in Example 3.
  • Certain acyltransferase polypeptides of the present invention exhibit acyltransferase activity that is at least about 1.6 fold greater than that of P. chrysogenum wild type acyltransferase (SEQ ID NO: 54).
  • acyltransferase polypeptides of the present invention may exhibit acyltransferase activity that is at least about 2 fold, sometimes at least about 5 fold, and in some embodiments at least about 10 fold or 20 fold greater up to about 30 fold or 50 fold greater than that of P. chrysogenum wild type acyltransferase.
  • the present invention provides acyltransferase polypeptides that are encoded by a polynucleotide that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 52.
  • nucleic acid hybridization experiments such as Southern and Northern hybridizations
  • Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters.
  • An extensive guide to the hybridization of nucleic acids is found in Tijessen (1993) "Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes," Part I, Chapter 2 (Elsevier, New York).
  • highly stringent hybridization and/or wash conditions are generally selected to be about 5 0 C or less lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH (as noted below, highly stringent conditions can also be referred to in comparative terms).
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • Stringent and highly stringent hybridization and wash conditions can be readily determined empirically for any nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, such as formamide, in the hybridization or wash), until a selected set of criteria is met.
  • the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, such as formamide, in the hybridization or wash), until a selected set of criteria is met.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 0 C, with the hybridization being carried out overnight.
  • An example of stringent wash conditions are 0.2x SSC wash at 65 0 C for 15 minutes (see Sambrook, et al., "Molecular Cloning - A Laboratory Manual” (1989) Cold Spring Harbor laboratory (Cold Spring Harbor, New York) for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example low stringency wash is 2x SSC at 4O 0 C for 15 minutes.
  • acyltransferase polypeptides of the present invention include those having an amino acid sequence corresponding to SEQ ID NOS: 2 (H1), 4 (H2), 6 (H3), 8 (H4), 10 (H5), 12 (H6), 14 (H7), 16 (H14), 18 (H25), 20 (H26), 22 (H27), 24 (H28), 26 (H29), 28 (H30), 30 (H31), 32 (H32), 34 (H33), 36 (H34), 38 (H35), 40 (H36), 42 (H37), 44 (H38), 46 (H40), 48 (H41), 50 (H42), and 52 (H43).
  • the acyltransf erase activities for these polypeptides are described in Example 3.
  • the present invention also provides acyltransferase polypeptides that are variants of these polypeptides having a substitution, deletion, and/or insertion of one to six amino acid residues.
  • variant refers to a sequence that has a high percent identity with respect to the reference sequence.
  • Variant acyltransferase polypeptides of the present invention may be naturally occurring or non-naturally occurring.
  • variant acyltransferase polypeptides may have a substitution, deletion, and/or insertion of one to six amino acid residues.
  • Such substitution, deletion and/or insertion may occur at more than one site in the polypeptide and may occur at the N-terminal and/or C-terminal end of the polypeptide as well as at one or more sites internal to the polypeptide.
  • Variant polypeptides encompassed by the present invention have acyltransferase activity.
  • Conservative variants can be readily generated by making conservative substitutions such as those within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagines), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (glycine, alanine, serine, threonine, proline, cysteine and methionine).
  • Variants of the acyltransferase polypeptides of the present invention may also be generated using mutagenesis and directed evolution methods that are well known to those having ordinary skill in the art. Libraries created by such methods can be screened for variants having acyltransferase activity as measured using the assay described in Example 3.
  • the present invention is directed to a fragment of an AT polypeptide of the present invention that exhibits AT activity in the assay described in Example 3. Fragments can be readily prepared using commercially available exonucleases and endonucleases according to known methods. As used herein, the term "fragment" refers to a polypeptide having a deletion of 1 to 15 amino acid residues from either or both the carboxy and/or amino terminus. In specific embodiments, the deletion is of 1 to 10 amino acid residues, and in some instances 1 to 5 amino acid residues.
  • AT fragments of the present invention have AT activity that is at least 1.6 fold greater than that of wild type P. chrysogenum AT activity using the assay described in Example 3. In some embodiments, AT fragments of the present invention have AT activity that is at least 2 fold, and typically up to about 30 fold greater than that of wild type P. chrysogenum AT activity in the assay of Example 3.
  • the present invention provides polynucleotides that encode acyltransf erase polypeptides of the present invention.
  • the present invention provides an isolated polynucleotide that encodes a polypeptide having acyltransferase activity, wherein the isolated polypeptide is selected from the group consisting of:
  • a polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 52 (d) a polypeptide having an amino acid sequence that is at least 96% identical to SEQ ID NO: 54 and having at least one residue selected from the group consisting of Arg at position 97, VaI at position 221 , Pro at position 251 , GIu at position 254, Ne at position 259, GIy at position 277, GIu at position 291, and GIy at position 299; and (e) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 42 and SEQ ID NO: 52.
  • Specific acyltransferase polynucleotides of the present invention include those having an polynucleotides sequence corresponding to SEQ ID NO: 1 (H1), 3 (H2), 5 (H3), 7 (H4), 9 (H5), 11 (H6), 13 (H7), 15 (H14), 17 (H25), 19 (H26), 21 (H27), 23 (H28), 25 (H29), 27 (H30), 29 (H31), 31 (H32), 33 (H33), 35 (H34), 37 (H35), 39 (H36), 41 (H37), 43 (H38), 45 (H40), 47 (H41), 50 (H41), and 51 (H43).
  • SEQ ID NOS: 13 and 15 are polynucleotide variants that encode the same acyltransferase polypeptide (i.e., SEQ ID NOS: 14 and 16 are the same).
  • SEQ ID NOS: 14 and 16 are polynucleotide variants that encode the same acyltransferase polypeptide.
  • each codon in a nucleic acid can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in any described sequence.
  • Acyltransferase polynucleotides may be codon optimized for expression in a particular host organism by modifying the polynucleotides to conform to the optimum codon usage of the desired host organism.
  • Polynucleotides of the present invention can be prepared using methods that are well known in the art. See, e.g., Carruthers, et al., Cold Spring Harbor Svmp. Quant. Biol.. 47:411-418 (1982) and Adams, et al., J. Am. Chem. Soc. 105:661 (1983). Typically, oligonucleotides of up to about 100 bases are individually synthesized, then enzymatically or chemically ligated together to form the desired sequence.
  • the present invention provides an expression cassette comprising an acyltransferase polynucleotide of the present invention operatively linked to a regulatory sequence, such as a promoter.
  • Expression cassettes of the present invention provide for the genetic transfer of acyltransferase polynucleotides, as well as expression and production of the acyltransferase polypeptides that they encode.
  • the regulatory sequences of the construct operate to drive transcription and translation of the acyltransferase polynucleotides.
  • the term "construct” refers herein to a single- or double-stranded nucleic acid molecule made up of DNA, RNA, synthetic analogues thereof, or a combination of two or more of these.
  • the nucleic acid construct may optionally contain other nucleic acid segments that may provide other functions useful in genetic transfer and/or expression, such as, for example, selectable marker genes.
  • the nucleic acid construct may be derived from a number of different sources, including bacterial, fungal, and plant. Suitable fungal sources include Aspergillus, and Penicillium (e.g., Penicillium chrysogenum).
  • the nucleic acid construct may be in the form of a vector that is suitable for manipulation, transformation, and/or expression of the acyltransferase polynucleotides of the present invention.
  • the nucleic acid construct may be selected and/or designed in accordance with the characteristics of the desired host organism into which it is to be introduced.
  • the construct may be autonomously replicating, i.e., it may exist normally as an extrachromosomal entity, e.g., a plasmid.
  • the nucleic construct may be a molecule which, when introduced into a host cell, is integrated into the genome.
  • Suitable regulatory sequences employed in the practice of the present invention may be any polynucleotide that exhibits appropriate activity in the host cell desired, and may be natural or synthetic in origin. Promoters that are suitable for use in filamentous funal host cells include, for example, the Aspergillus nidulans trpC promoter (Yelton, M.M., et al., Proc. Natl. Acad. Sci. USA, 81 :1470-1474 (1984); Mullaney, E.J. et al., MoI. Gen.
  • Suitable promoters include those from the penicillin biosynthetic pathway itself, such as the Penicillium chrysogenum pcbC (IPNS) promoter (Barredo, J. L., et al., MoI. Gen. Genet., 216(1 ):91-98 (1989)) or the Penicillium chrysogenum penDE (AT) promoter (Diez, B., et al., MoI.
  • IPNS Penicillium chrysogenum pcbC
  • IPNS Penicillium chrysogenum pcbC
  • AT Penicillium chrysogenum penDE
  • Nucleic acid constructs of the present invention may also comprise a selectable marker, e.g., a polynucleotide encoding a marker that facilitates the selection of the cell that contains the nucleic acid construct.
  • a selectable marker e.g., a polynucleotide encoding a marker that facilitates the selection of the cell that contains the nucleic acid construct.
  • selectable markers include kanamycin resistance genes, chloramphenicol resistance genes, as well as other antibiotic resistance-conferring genes.
  • Preferred selectable markers for use in filamentous fungi include genes encoding acetamidease (Corrick, CM.
  • genes that are useful as selection markers are those that encode factors for auxotrophic complementation, such as, for example, PyrG (Cantoral, J. M., et al., Nucleic Acids Res., 16(16):8177 (1988)), ArgB (Upshall, A., et al., MoI. Gen. Genet.. 204(2):349-54 (1986)), niaD (Johnstone, I.L, et al., Gene, 90(2):181- 92 (1990)), and TrpC (Penalva, M.A., Nucleic Acids Res., 15(41:1874 (1987)).
  • nucleic acid constructs containing the ampicillin resistance gene are less useful when beta-lactam production is the reaction of interest because the gene encodes beta-lactamase, which, if expressed, will degrade the beta-lactam.
  • a marker such as phleomycin resistance gene (phlR) can be used in Penicillium species when beta-lactam production is the reaction of interest.
  • Nucleic acid constructs of the present invention may also contain a targeting signal or secretory signal sequence to direct expression of the acyltransferase polypeptide to a desired location within the host cell or into the fermentation media.
  • the targeting signal and/or secretory signal sequence is joined to the acyltransferase polynucleotide so that both are in the same reading frame.
  • These signal sequences may be native to the host cell, or they may be of synthetic or foreign origin. Where the host organism is a filamentous fungus, the signal peptide may be derived from a fungal enzyme, for example, Aspergillus spp. amylase.
  • the host cell into which the construct is introduced may be any cell having the desired properties.
  • it may be a cell that can produce large amounts of the acyltransferase polypeptide of the present invention. It may also be a host cell that is capable of producing a beta-lactam compound, i.e. a host cell that contains the machinery to produce beta-lactam compounds.
  • the host cell is a bacterium, a yeast or a filamentous fungus.
  • a filamentous fungus may be, for example, Aspergillus spp. or Penicillium chyrsogenum. The use of Aspergillus spp.
  • the transformed host cell containing the construct is cultured in a suitable nutrient medium under conditions permitting the expression of the polypeptide, after which the polypeptide may be recovered. Recovery may be accomplished, for example, by separation of the host cells from the medium by centrifugation or filtration. Where the polypeptide is produced intracellular, the cells are recovered from the fermentation media by centrifugation or filtration and the cells are homogenized to release the polypeptide.
  • Proteinaceous components may then be precipitated from the media or supernatant by means of a salt (e.g., ammonium sulfate) and purified by a variety of chromatographic and/or electrophoretic procedures (e.g., ion exchange chromatography, gel filtration chromatography, isoelectric focusing, affinity chromatography, and the like). Recovery techniques are often dependent on the characteristics of the particular polypeptide.
  • the polypeptide may then be further used.
  • the transformed host cell containing the construct according to the invention is cultured in a suitable nutrient medium under conditions permitting the expression of a beta-lactam compound of interest.
  • the present invention provides a process for the production of a beta-lactam compound comprising culturing the transformed host cell containing the construct according to the invention under conditions conducive to the production of an N-acylated beta-lactam compound; optionally deacylating the produced N-acylated beta- lactam compound to produce the corresponding N-deacylated compound; and recovering the N-acylated or N-deacylated beta-lactam compound.
  • the skilled person will immediately recognize that the nature of the beta-lactam compound that is produced depends on the transformed host that is used. If the transformed host containing the construct according to the invention is a genetically modified P.
  • the N-deacylated beta-lactam compound is 7-ADCA.
  • the transformed host cell containing the construct according to the invention is an Acremonium chrysogenum strain naturally producing expandase, the N- deacylated beta-lactam compound is 7-ACA.
  • N-acyl side chain precursor to be used in the process performed under conditions conducive to the production of a N-acylated beta-lactam compound preferably is adipic acid or a salt thereof.
  • other side chain precursors may be used, for instance those mentioned in international applications WO95/04148, WO95/04149, WO98/48034 or WO 98/48035.
  • the various polynucleotide elements may be manipulated such that they are in the proper orientation and in the proper reading frame. Toward this end, adapters or linkers may be employed to join the polynucleotide elements. Likewise, various known procedures may be employed to introduce convenient restriction sites, remove superfluous nucleotides, remove restriction sites, and the like.
  • Polynucleotide sequences of the present invention can be used to identify and/or isolate corresponding sequences from other organisms, such as other fungi. Methods known in the art, such as PCR, hybridization, and the like, can be used to identify sequences based on their degree of sequence identity to the sequences of the present invention, and sequences isolated based on their sequence identity to sequences set forth herein are encompassed by the present invention. Appropriate methods for such identification are known in the art and described in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). For example, fragments of the nucleotide sequences disclosed herein may be used as probes.
  • Such probes would be capable of specifically hybridizing to corresponding nucleotide sequences as well as messenger RNAs and could be used, for example, to distinguish single base polymorphisms in different candidate sequences in an SSCP assay.
  • probes include sequences that differ between the candidate sequences.
  • Such probes may be used to amplify corresponding acyltransf erase sequences from a chosen organism by PCR; this technique may be used to isolate further sequences from an organism or as a diagnostic assay to determine the presence or expression level of coding sequences in an organism.
  • Such genes may be cloned by culturing of the organisms, extracting the total RNA, purifying the mRNA and constructing a cDNA library using reverse transcriptase. Standard procedures for DNA techniques are described in, for example, Sambrook et al. (Sambrook, J., Fritsch, E. F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor
  • the genes can be isolated by designing and synthesizing PCR primers that anneal to the 5'and 3' termini of the acyltransferase genes, amplifying the specific gene from the cDNA library and cloning in an expression vector.
  • the present example describes a method by which acyltransferase genes of known sequence may be synthesized starting from short oligonucleotides. The assembly of such synthetic genes has been widely described (Stemmer WP et al. 1995, Gene 164: 49; Jayaraman K et al. 1989, Nucleic Acids Res. 17(11): 4403; Holowachuk EW et al. 1995, PCR Methods Appl. 4(5): 299; Prodromou C et al. 1992, Protein Eng. 5(8): 827).
  • the oligonucleotides are mixed together at a uniform concentration of 250 ⁇ M.
  • the mixture is diluted 100-fold in 20 ⁇ l PCR mix containing 1OmM Tris-HCI pH 9.0/2.2 rtiM MgCI 2 /50 mM KCI/ 0.1% Triton X-100 [1x Cloned Pfu buffer (Stratagene, La JoIIa, CA, USA)]/0.2 mM each deoxynucleotide (Stratagene, La JoIIa, CA, USA)/1 u of Taq DNA polymerase (Promega, Madison, Wl, USA)/0.02 u of Pfu DNA polymerase (Stratagene, La JoIIa, CA, USA).
  • thermocycler MJ Research PTC-150 minicycler (MJ Research, Inc., Reno, NV, USA) set to the following program of 55 cycles at 94°C for 30 seconds, 52°C for 30 seconds and 72°C for 30 seconds.
  • the resulting gene assembly mixture is diluted 40-fold in 100 ⁇ l PCR mix containing 1OmM Tris-HCI pH 9.0/2.2 mM MgCI 2 /50 mM KCI/ 0.1% Triton X-100 [1x Cloned Pfu buffer (Stratagene, La JoIIa, CA, USA)]/0.2 mM each deoxynucleotide (Stratagene, La JoIIa, CA, USA)/5 u of Taq DNA polymerase (Promega, Madison, Wl, USA)/0.1 u of Pfu DNA polymerase (Stratagene, La JoIIa, CA, USA)/2 outside primers at a concentration of 1 ⁇ M.
  • the 2 outside primers can be the same as the two oligonucleotides representing the 5' ends of the plus and minus strand, in this example the outside primer that anneals to the 5'-end of the plus strand adds an Ndel recognition site (the ATG of the second half of the recognition sequence overlaps the initiation codon of the acyltransf erase gene) and the outside primer that anneals to the 5'-end of the minus strand adds an Notl recognition site.
  • the gene is amplified in a thermocycler [MJ Research PTC-150 minicycler (MJ Research, Inc., Reno, NV, USA)] set to the following program of 23 cycles at 94°C for 30 seconds, 50 0 C for 30 seconds and 72°C for 60 seconds.
  • the final gene is purified using the QIAquick PCR Purification Kit (USA- QIAGEN Inc., Valencia, CA, USA), digested with Ndel and Notl (New England Biolabs, Beverly, MA, USA) and the resulting 1.0-kb fragment is purified using the QIAquick Gel Extraction kit (USA-QIAGEN Inc., Valencia, CA, USA).
  • the synthesized penDE gene from Penicillium chrysogenum ATCC9480 can be cloned into an appropriate expression vector.
  • acyltransferase genes described herein may be constructed using a similar procedure by introduction of the relevant changes to the oligonucleotide sequences (reflecting the changes in sequence compared to the penDE gene from Penicillium chrysogenum ATCC9480).
  • Plasmid pET24B is one of the pET series of vectors created by Studier (Moffatt, B.A. et al. 1986, J. MoI. Biol. 189:113) and further developed by Novagen (Novagen, Inc., Madison, Wl, USA) to facilitate cloning, detection, and purification of recombinant proteins in E.coli. These vectors typically carry the colicin E1 replicon of pBR322 and confer resistance to ampicillin or kanamycin. There are two general categories of vectors in the pET series, transcription and translation.
  • the translation vectors which contain a highly efficient ribosomal binding site from phage T7, are designed to express a gene without its own ribosomal binding site.
  • transcription vectors are designed for expressing target gene with its own prokaryote's ribosomal binding site.
  • plasmid pET24B a translation vector for cloning of the acyltransferase gene from Penicillium chrysogenum ATCC9480 illustrates how expression of an active acyltransferase enzyme may be performed.
  • the Multiple Cloning Site (MCS) of pET24B comprises both an Ndel and Notl recognition sequences, allowing the directional cloning of the synthesized acyltransferase gene.
  • pET24B is digested with Ndel and Notl (New England Biolabs, Beverly, MA, USA) and purified using the QIAquick Gel Extraction kit (USA-QIAGEN Inc., Valencia, CA, USA) generating an appropriate expression vector.
  • the complementary cohesive termini of the two fragments (pET24B vector and acyltransferase insert) are ligated using a T4 DNA ligase kit (New England Biolabs, Beverly, MA, USA).
  • pET-penDE Pc Figure 1
  • replication and expression of the gene can be confirmed by transforming the recombinant plasmid into E.coli BL21 cells (Novagen, Inc., Madison, Wl, USA) and then selecting cells based upon the expression of antibiotic resistant gene carried on the plasmid.
  • kanamycin is used as a selection marker for cells, containing an intact and functional plasmid, which may or may not carry the acyltransferase gene.
  • a small number of clones are picked, grown in liquid cultures and DNA is prepared using a QIAprep Spin Miniprep Kit (USA-QIAGEN Inc., Valencia, CA, USA).
  • the DNA is sequenced on an ABI 3700 DNA Analyzer (Applied Biosystems, Inc, Foster City, CA, USA) using pET sequencing primers (Novagen, Inc., Madison, Wl, USA) and one clone with the correct acyltransferase sequence is selected. Characterization of acyltransferase activity is described in Example 2.
  • Plasmid pET-penDE Pc is transformed into chemically competent E.coli
  • BL21(DE3) cells Novagen, Inc., Madison, Wl, USA
  • LB media agar EM Science, Gibbstown, NJ, USA
  • glucose Sigma, St. Louis, MO, USA
  • chloramphenicol (30ug/mL)
  • Individual colonies formed on the plate are picked and used to inoculate 2xYT media (EM Science, Gibbstown, NJ, USA) containing 0.5% glucose and chloramphenicol.
  • the inclusion of glucose in the media is important to reduce premature gene expression, which may be detrimental to cell growth.
  • the culture is grown overnight in a shaking incubator at 37°C.
  • Expression of the acyltransferase gene is induced by the addition of 0.2mM isopropyl- ⁇ -D- thiogalactopyranoside (IPTG) (Sigma, St. Louis, MO, USA), and the culture is further incubated for 16h, shaking at 28°C. Cells are harvested by centrifugation at 4000 rpm at 4°C and washed at least three times with 5mM morpholine (Sigma, St.
  • This assay is used to evaluate acyltransf erase specific activity that converts 6- APA and Ad-CoA substrates into Ad-6-APA product.
  • Separate stock solutions of 1.5mM 6-APA, 5OmM DTT, and 0.6mM Ad-CoA are prepared freshly by dissolving each in 5mM morpholine buffer, pH 7.5. 6-APA and DTT are available from Sigma (St. Louis, MO, USA).
  • Adipoyl-Coenzyme A is obtained as follows: Adipoyl chloride (0.14 ml; 6.95 mmol) was added to a well-stirred and ice-water cooled suspension of Co-enzyme A sodium salt (400 mg; 0.48 mmol) in acetone (60 ml) + water (0.6ml) + 0.2M KHCO 3 (to raise the pH) under an atmosphere of nitrogen. The pH of the reaction contents was brought to -7.7 and stirred further for about 60 min. Thereafter acetone was removed partially under reduced pressure, the product dissolved in cold water and this was freeze-dried. Finally the product was subjected to ultrafiltration. The yield was 0.9 g. Assay 70%. The water used was treated with a stream of nitrogen gas to avoid oxidation of co-enzyme A.
  • 8ml_ of 1.5mM 6-APA, 4ml_ of 0.6 rtiM Ad-CoA, and 4ml_ of 5OmM DTT are combined to create a master solution of 0.75mM 6-APA, 0.15mM Ad-CoA, 12.5mM DTT, 5mM morpholine buffer, pH7.5.
  • 300 ⁇ L of 5mM morpholine buffer pH 7.5 is added to the solution.
  • 155 ⁇ L of 5mM morpholine buffer is transferred into a tube or a well of the 96-well plate.
  • the sample is filtered, for example, through a Whatman custom filter plate (1 micron pore size) (Whatman Inc., Clifton, NJ, USA) and the filtrate is collected in a Nunc V-bottom polystyrene microtiter plate (Nunc, Rochester, NY, USA). The plate is sealed with regular aluminum foil. Ad-6-APA product can be detected using mass spectrometry. Electrospray ionization mass spectrometry (ESI/MS) analyses may be performed on a Quattro Ultima triple-quadruple mass spectrometer (Micromass, Manchester, UK) with an electrospray ion source. Typically, samples are dissolved in methanol/water (1 :1) as described in example 2.
  • ESI/MS Electrospray ionization mass spectrometry
  • Adipoyl-6-APA will yield intense deprotonated molecules ([M-H]- in the negative-ion mode.
  • the capillary voltage is 3.OkV
  • source temperature is 12O 0 C
  • desolvation temperature is 25O 0 C.
  • Adipoyl-6-APA is monitored by mass transition 343.0 to 265.0, under cone energy of 15V, collision energy of 15eV.
  • Ad-6-ApA can be quantified by comparing the area of the sample peak to a standard curve. The standard curve is computed by injecting ad-6-APA samples of known concentration and measuring the peak area.
  • FIA flow injection
  • H04 (SEQ ID NO: 8) H05 (SEQ ID NO: 10)
  • H26 (SEQ ID NO: 20) H27 (SEQ ID NO: 22)
  • H30 (SEQ ID NO: 28) H31 (SEQ ID NO: 30)
  • H36 (SEQ ID NO: 40) H37 (SEQ ID NO: 42)
  • H42 (SEQ ID NO: 50) H43 (SEQ ID NO: 52)

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Abstract

La présente invention concerne de nouveaux polypeptides d'acyltransférase, ainsi que les polynucléotides les codant, qui sont particulièrement utilisés dans la production d'intermédiaires de β-lactam et de composés antibiotiques possédant des chaînes latérales d'adipoyle. Cette invention a aussi pour objet des vecteurs, des cellules hôtes et des méthodes de conception et d'utilisation desdits nouveaux polypeptides d'acyltransférase.
EP06708501A 2005-02-24 2006-02-24 Mutants de acyltransferase de isopenicillin n Withdrawn EP1851309A2 (fr)

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